This invention relates to compositions based on xcex22 microglobulin, and the use of such compositions in immunological methods pertaining to the targeting of proteins to cell surfaces. The disclosed compositions and methods have particular application to vaccination and tumor therapy.
MHC I and Activation of Cytotoxic T-cells
The beta-2 microglobulin (xcex22m) protein is a component of the class I major histocompatibility complex (MHC I). MHC I is formed by the association of xcex22m and an alpha protein (also known as the xe2x80x9cheavyxe2x80x9d chain), which comprises three domains, a1, a2 and a3. MHC I is found on the surface of most types of nucleated cells, where it presents antigens derived from proteins synthesized in the cytosol to CD8+ T-cells. Two signals are required for activation of naive CD8+ T-cells. The first signal is provided by the interaction of the T-cell receptor (TCR) with the MHC I-antigen complex on the antigen-presenting cell surface. The second signal is generated by the interaction of a ligand on the antigen-presenting cell (APC) with a second receptor present on the T-cell surface. This second signal is termed co-stimulation, and the APC ligand is often referred to as a co-stimulatory molecule. The best characterized co-stimulatory molecules on APCs are the structurally related glycoproteins B7.1 (CD80) and B7.2 (CD86) which interact with the CD28 receptor on the T-cell surface. Activation of CD8+ T-cells by these two signals leads to the proliferation of antigen-specific cytotoxic T-cells, which recognize and destroy cells presenting the signaling antigen. These cytotoxic T-cells play an important role in the immunological defense against intracellular pathogens such as viruses, as well as tumors. A detailed presentation of the immunological basis of the cytotoxic T-cell response can be found in Janeway and Travers (Immunobiology: the immune system in health and disease, Current Biology Ltd./Garland Publishing, Inc. New York, 1997).
The failure of an exogenous (non-self) antigen to stimulate a cytotoxic T-cell response can result from a block in the above-described cytotoxic T-cell activation pathway at one of many points (see Ploegh, 1998, Science 280:248-53). Failure of the cytotoxic T-cell activation pathway is of great significance in two particular areas of medicine: vaccination and tumor immunology.
Cytotoxic T-cells and Vaccination
Vaccine technology has focused in recent years on sub-unit vaccines. Sub-unit vaccines comprise isolated pathogen components, such as viral capsid or envelopes, or synthetic peptides that mimic an antigenic determinant of a pathogen-related protein. For example, U.S. Pat. No. 4,974,168 describes leukemia associated immunogens that are peptides based on envelope proteins of a leukemia-associated virus. However, while sub-unit vaccines can stimulate CD4+ helper T-cells (which play a key role in humoral immunity), attempts to stimulate CD8+ cytotoxic T-cells in vivo with such vaccines have been largely unsuccessful. It has been postulated that the reason for this is the inability of the exogenously administered vaccine peptide to associate with the MHC I molecules on the cell surface (Liu, 1997, Proc. Natl. Acad Sci. USA 94:10496-8). In other words, the block in the cytotoxic T-cell activation pathway occurs at the stage where the antigen is loaded into the MHC I molecule.
One proposed solution to this problem is to combine the antigenic peptide with a molecule that is readily taken up into cells (reviewed by Liu, 1997, Proc. Natl. Acad Sci. USA 94:10496-8). Thus, this strategy is based on getting the antigen into the cytosol so that it can join the normal pathway by which antigens are processed for presentation by MHC I. In contrast, Rock et al. (J. Immunol. 150:1244-52, 1993) adopted a strategy of enhancing the binding of the vaccine peptide to MHC I already present on the cell surface. Rock et al. (J. Immunol. 150:1244-52, 1993) report that the administration of exogenous purified xcex22m along with the vaccine peptide produces enhanced loading of the peptide onto MHC I in vivo and thereby stimulates a cytotoxic T-cell response against the peptide. The use of exogenous xcex22m as a vaccine adjuvant is also described in U.S. Pat. No. 5,733,550 (to Rock et al.), which is incorporated herein by reference.
Tumor Cells and Immune System Evasion
Tumor cell immunity is primarily cell-mediated, involving both CD8+ cytotoxic T-cells and CD4+ helper T-cells. However, despite the fact that tumor cells express tumor-specific proteins that are not recognized as self-antigens by the immune system, they often escape recognition by the immune system. A number of factors may contribute to the ability of tumor cells to evade immune recognition, including the down-regulation of expression of co-stimulatory proteins. TCR stimulation in the absence of co-stimulatory molecules can result in failure to activate the T-cell and the induction of clonal anergy. Thus, down-regulation of co-stimulatory proteins in tumor cells prevents normal activation of T-cells that do bind to tumor antigens on the cell surface, permitting the tumor cell to escape recognition.
Several research groups have attempted to address this issue by removing tumor cells from a patient, providing exogenous co-stimulatory molecules on the surface of the removed tumor cells and then reintroducing the tumor cells to the patient so that immune recognition can occur. For example, European patent application number 96302009.4 describes a method by which tumor cells are removed from a patient, transfected to express both B7 and CD2 (a co-receptor involved in T-cell adhesion and activation) on the tumor cell surface, and then reintroduced to the patient. The reintroduced cells are reported to stimulate a broad immunological response against both the reintroduced transfected tumor cells and the non-transfected tumor cells within the patient""s body, resulting in tumor regression.
Adopting an alternative approach to this problem, Gerstmayer et al. (J. Immunol. 158:4584-90, 1997) describe a chimeric B7-antibody protein, in which the antibody is specific for the erbB2 proto-oncogene product. This chimeric molecule localizes specifically on the surface of erbB2 expressing tumor cells, and presents the B7 co-stimulatory molecule to cytotoxic T-cells, resulting in enhanced proliferation of cytotoxic T-cells. Gerstmayer et al. (J. Immunol. 158:4584-90, 1997) thus propose that fusion proteins comprising an anti-tumor antibody and a co-stimulatory molecule could be useful as tumor immunotherapeutics. However, this approach would require prior knowledge and characterization of tumor-specific antigens expressed on the tumor cells of each individual patient, and the use of an antibody specific for that particular type of tumor cell.
The present invention employs various forms of beta-2 microglobulin to address the problems associated with failure of the cytotoxic T-cell activation pathway in both vaccination and tumor therapy. The invention also provides compositions and methods based on xcex22m that are broadly applicable to achieve expression of any desired target protein on the surface of any mammalian cell.
In one embodiment, the invention provides fusion proteins comprising a first amino acid sequence and a second amino acid sequence, wherein the second amino acid sequence is a xcex22-microglobulin. In particular applications, the first amino acid sequence may be a co-stimulatory protein, such as B7.1 or B7.2, or another protein having immunological activity, such as a cytokine, an integrin or a cellular adhesion molecule. Examples of such proteins include interleukins (e.g., IL-2, IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphocyte function-associated proteins (e.g., LFA-1, LFA-3) and intercellular adhesion molecules (e,g., ICAM-1, ICAM-2). In other embodiments, the first amino acid sequence of the fusion protein may be any protein that is desired to be expressed on the surface of a cell. It is shown that these fusion proteins are an effective way to target a desired protein, such as B7, to the outer membrane of a cell. (xe2x80x9cB7xe2x80x9d is used generically to refer to either B7.1 or B7.2).
With respect to tumor therapy, it is shown that expressing on the surface of a tumor cell a fusion protein comprising a xcex22m joined to a co-stimulatory protein can significantly increase the immune response of an animal to the tumor cell. In one example, a fusion protein comprising hxcex22m joined to the co-stimulatory protein B7 (and termed B7-xcex22m) is targeted to the surface of tumor cells, such that the tumor cells present the B7-xcex22m fusion protein to T-cells. These cells are then attenuated and introduced into mice. T-cells removed from these mice were shown to be significantly more active against the same type of tumor cells than equivalent cells from mice treated with tumor cells presenting xcex22m only.
The xcex22m fusion proteins provided by the invention have wide application in that they are useful to target any desired protein to the outer membrane of a cell. These fusion proteins may be targeted to the surface of a cell in a number of ways. In one approach, cells that express MHC I are simply incubated with a preparation of the fusion protein, resulting in the incorporation of the fusion protein onto the cell surface. Alternatively, the fusion protein may be introduced into the cell so that it is incorporated into the MHC I pathway. In another approach, a nucleic acid molecule encoding the fusion protein is introduced into a cell by transformation. Expression of this nucleic acid molecule results in the fusion protein being produced within the cell and exported to the cell membrane. Where the fusion protein is to be introduced into the cytosol for export to the outer membrane, or where it will be expressed by a nucleic acid molecule within the cell, it is desirable to include a signal peptide at the N-terminus of the fusion peptide so that the fusion protein is transported to the outer membrane of the cell. The xcex22m signal peptide may be used for this purpose. In all of these approaches, the result is that the xcex22m fusion protein is presented on the surface of the cell.
In one embodiment, the invention includes nucleic acid molecules encoding the disclosed fusion proteins, as well as nucleic acid vectors comprising such nucleic acid molecules. Transgenic cells comprising these nucleic acid molecules are also provided by the invention. Methods of expressing a xcex22m fusion protein on the surface of a cell are provided by the invention. Such methods include contacting a cell with a fusion protein comprising a first amino acid sequence and a second amino acid sequence wherein the second amino acid sequence is a xcex22m. An alternative method provided by the invention comprises transforming the cell with a nucleic acid molecule encoding such a fusion protein.
The invention further provides methods of enhancing the immune response of an animal to an antigen presented on the surface of a cell. Such methods comprise providing a xcex22m fusion protein on the surface of the cell and administering the cell to the animal. In such applications, the fusion protein preferably comprises xcex22m fused to a co-stimulatory protein, such as B7, or another protein having immunological activity. Expressing the xcex22m fusion protein on the surface of the cell may be accomplished by contacting the cell with the fusion protein, or transforming the cell with a nucleic acid molecule encoding the protein. These methods may be applied to the treatment of tumors; in such treatments, the antigen against which an enhanced immune response is desired is a tumor antigen, and the cell bearing the antigen is a tumor cell. The tumor cell may be removed from the body of a mammal having a tumor, or may be derived from an in vitro propagated tumor cell line. The xcex22m fusion protein is introduced to the tumor cell (e.g., by incubation of the tumor cell with the protein, or by transformation of the tumor cell with a nucleic acid encoding the fusion protein), such that the tumor cell presents the fusion protein on its surface. The tumor cell carrying the fusion protein is then administered to a mammal. In certain embodiments, the tumor cell may be attenuated prior to being administered to the mammal; such attenuation may be accomplished using standard means such as radiation, heat or chemical treatment. Once in the body of the mammal, the combination of tumor antigens and the xcex22m-fusion protein on the surface of the tumor cells are recognized by CD8+ T-cells, resulting in T-cell activation, proliferation and thereby an enhanced cytolytic T-cell response against both the introduced tumor cells and other tumor cells in the mammal that express the same tumor antigen.
The present invention also provides modified human xcex22m (hxcex22m) proteins having an enhanced affinity for MHC I. Such proteins are shown to bind to the alpha chain of MHC I with higher affinity than wild-type hxcex22m and to enhance T-cell recognition of APCs bearing the modified hxcex22m. In particular embodiments, the modified hxcex22m proteins have a valine residue at position 55 in place of the serine residue that is found in the mature form of naturally occurring (i.e., wild-type) hxcex22m . Such modified hxcex22m proteins are referred to as hxcex22m S55V.
hxcex22m S55V is useful as a vaccine adjuvant in place of wild-type hxcex22m. Thus, one aspect of the invention is a vaccine preparation comprising at least one antigen and hxcex22m S55V. hxcex22m S55V may also be utilized in place of wild-type hxcex22m in the fusion proteins discussed above. Additionally, xcex22m fusion proteins may also be employed in such vaccine preparations, either using a wild-type xcex22m or, in the case of hxcex22m, hxcex22m S55V.
The nucleic and amino acid sequences listed in the Sequence Listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
Seq. I.D. No. 1 shows the amino acid sequence of wild-type (naturally occurring) hxcex22m.
Seq. I.D. No. 2 shows the amino acid sequence of the B7-xcex22m fusion protein (comprising the B7.2 co-stimulatory molecule).
Seq. I.D. No. 3 shows the amino acid sequence of the B7-xcex22m fusion protein having the xcex22m signal sequence joined to the N-terminal of the B7 domain.
Seq. I.D. Nos. 4-7 show primers used to construct hxcex22m S55V.
Seq. I.D. Nos. 8 and 9 show primers used to amplify the B7.2 protein.
Seq. I.D. No. 10 shows the amino acid sequence of mature hxcex22m S55V.
Seq. I.D. Nos. 11 and 12 show the amino acid linker sequences that can be used between the two domains of a fusion protien.
Seq. I.D. Nos. 13 and 14 show amino acid sequences of signal peptides that can be used to direct the expression of a protein in a cell.
Seq. I.D. No. 15 shows the amino acid sequences for a c-myc tag.
Seq. I.D. Nos. 16-20 show the amino acid sequences for peptides used in the HLA stabilization assay.